The long intergenic noncoding RNA landscape of human lymphocytes highlights the regulation of T cell differentiation by linc-MAF-4.

Long noncoding RNAs are emerging as important regulators of cellular functions, but little is known of their role in the human immune system. Here we investigated long intergenic noncoding RNAs (lincRNAs) in 13 subsets of T lymphocytes and B lymphocytes by next-generation sequencing-based RNA sequencing (RNA-seq analysis) and de novo transcriptome reconstruction. We identified over 500 previously unknown lincRNAs and described lincRNA signatures. Expression of linc-MAF-4, a chromatin-associated lincRNA specific to the TH1 subset of helper T cells, was inversely correlated with expression of MAF, a TH2-associated transcription factor. Downregulation of linc-MAF-4 skewed T cell differentiation toward the TH2 phenotype. We identified a long-distance interaction between the genomic regions of the gene encoding linc-MAF-4 and MAF, where linc-MAF-4 associated with the chromatin modifiers LSD1 and EZH2; this suggested that linc-MAF-4 regulated MAF transcription through the recruitment of chromatin modifiers. Our results demonstrate a key role for lincRNA in T lymphocyte differentiation.

Clinical effectiveness of platelets in additive solution treated with two commercial pathogen-reduction technologies.

BACKGROUND: Two noninferiority, randomized, controlled trials were conducted in parallel comparing the safety and efficacy of platelets treated with Intercept or Mirasol pathogen-reduction technologies versus standard platelets. STUDY DESIGN AND METHODS: The primary endpoint was the percentage of hematology patients who developed World Health Organization Grade 2 or greater bleeding. A noninferiority margin of 11% was chosen based on expected Grade 2 or greater bleeding in 20% of controls. The study was closed for financial restrictions before reaching the planned sample size of 828 patients, and an intention-to-treat analysis was conducted on 424 evaluable patients. RESULTS: In the Intercept trial (113 treated vs. 115 control patients), the absolute risk difference in Grade 2 or greater bleeding was 6.1%, with an upper one-sided 97.5% confidence limit of 19.2%. The absolute risk difference in the Mirasol trial (99 treated vs. 97 control patients) was 4.1%, and the upper one-sided 97.5% confidence limit was 18.4%. Neither absolute risk difference was statistically significant. In both trials, posttransfusion platelet count increments were significantly lower in treated versus control patients. Mean blood component use in treated patients versus controls was 54% higher (95% confidence interval, 36%-74%; Intercept) and 34% higher (95% confidence interval, 16%-54%; Mirasol) for platelets and 23% higher (95% confidence interval, 8%-39%; Intercept) and 32% higher (95% confidence interval, 10%-57%; Mirasol) for red blood cells. Unexpected reactions and adverse events were not reported. Mortality did not differ significantly between treated and control patients. CONCLUSION: Although conclusions on noninferiority could not be drawn due to low statistical power, the study provides additional information on the safety and efficacy of pathogen-reduced platelets treated with two commercial pathogen-reduction technologies.

An update on methods for cryopreservation and thawing of hemopoietic stem cells.

The aim of this article is to review a number of variables that may affect the cryopreservation of minimally manipulated products containing allogeneic or autologous hemopoietic progenitor cells (HPC) used for transplantation, with particular reference to processing, type and addition of cryoprotectant, cell concentration, volume, freezing procedure, cooling rate, storage, thawing, and quality management. After defining final product's requirements in compliance with norms, laws and regulations, it is crucial to define the critical control points of the process. New approaches of processing were developed in the last few years such as automatic devices for volume reduction and high cell concentration in the frozen product. DMSO at 10% final concentration is still the most used cryoprotectant for HPC cryopreservation. Although controlled rate freezing is the recommended method for HPC cryopreservation, alternative methods may be used. Last generation vapor storage vessels ensure temperature stability better than older tanks. Their use may reduce risks of cross-contamination. Finally we review advantages and disadvantages of thawing procedures that may be carried out in the laboratory or at the patient's bedside.

HEA BeadChip™ technology in immunohematology.

Classic methods to determine human red blood cell (RBC) antigens are based on serologic testing. Thanks to increased knowledge of the molecular basis associated with many blood group antigens, it is currently possible to predict their presence or absence on the red cell membrane. Several molecular techniques have been developed to detect the most important allelic variations attributable to single nucleotide polymorphisms. The human erythrocyte antigen (HEA) BeadChip™ system manufactured by BioArray Solutions (Immucor, Warren, NJ) is one of the commercial DNA array platforms currently available to predict HEAs by DNA analysis. This technology provides a useful tool to increase the inventory of antigen-negative RBC units and prevent immunization of patients who require chronic transfusion by providing compatible RBC units based on matching by DNA testing.

Immunochip analyses identify a novel risk locus for primary biliary cirrhosis at 13q14, multiple independent associations at four established risk loci and epistasis between 1p31 and 7q32 risk variants.

To further characterize the genetic basis of primary biliary cirrhosis (PBC), we genotyped 2426 PBC patients and 5731 unaffected controls from three independent cohorts using a single nucleotide polymorphism (SNP) array (Immunochip) enriched for autoimmune disease risk loci. Meta-analysis of the genotype data sets identified a novel disease-associated locus near the TNFSF11 gene at 13q14, provided evidence for association at six additional immune-related loci not previously implicated in PBC and confirmed associations at 19 of 22 established risk loci. Results of conditional analyses also provided evidence for multiple independent association signals at four risk loci, with haplotype analyses suggesting independent SNP effects at the 2q32 and 16p13 loci, but complex haplotype driven effects at the 3q25 and 6p21 loci. By imputing classical HLA alleles from this data set, four class II alleles independently contributing to the association signal from this region were identified. Imputation of genotypes at the non-HLA loci also provided additional associations, but none with stronger effects than the genotyped variants. An epistatic interaction between the IL12RB2 risk locus at 1p31and the IRF5 risk locus at 7q32 was also identified and suggests a complementary effect of these loci in predisposing to disease. These data expand the repertoire of genes with potential roles in PBC pathogenesis that need to be explored by follow-up biological studies.

Mononuclear cells from a rare blood donor, after freezing under good manufacturing practice conditions, generate red blood cells that recapitulate the rare blood phenotype.

BACKGROUND: Cultured red blood cells (cRBCs) from cord blood (CB) have been proposed as transfusion products. Whether buffy coats discarded from blood donations (adult blood [AB]) may be used to generate cRBCs for transfusion has not been investigated. STUDY DESIGN AND METHODS: Erythroid progenitor cell content and numbers and blood group antigen profiles of erythroblasts (ERYs) and cRBCs generated in human erythroid massive amplification (HEMA) culture by CB (n = 7) and AB (n = 33, three females, three males, one AB with rare blood antigens cryopreserved using CB protocols) were compared. RESULTS: Variability was observed both in progenitor cell content (twofold) and number of ERYs generated (1 log) by CB and AB in HEMA. The average progenitor cell contents of the subset of AB and CB analyzed were similar. AB generated numbers of ERYs three times lower (p < 0.01) than CB in HEMA containing fetal bovine serum but similar to CB in HEMA containing human proteins. Female AB contained two times fewer (p < 0.05) erythroid progenitor cells but generated numbers of ERYs similar to those generated by male AB. Cryopreserved AB with a rare blood group phenotype and shipped to another laboratory generated great numbers of ERYs, 90% of which matured into cRBCs. Blood group antigen expression was consistent with the donor genotype for ERYs generated both by CB and AB but concordant with that of native RBCs only for cells derived from AB. CONCLUSION: Buffy coats from regular donors, including a donor with rare phenotypes stored under conditions established for CB, are not inferior to CB for the generation of cRBCs.

Identification of new autoantigens by protein array indicates a role for IL4 neutralization in autoimmune hepatitis.

Autoimmune hepatitis (AIH) is an unresolving inflammation of the liver of unknown cause. Diagnosis requires the exclusion of other conditions and the presence of characteristic features such as specific autoantibodies. Presently, these autoantibodies have relatively low sensitivity and specificity and are identified via immunostaining of cells or tissues; therefore, there is a diagnostic need for better and easy-to-assess markers. To identify new AIH-specific autoantigens, we developed a protein microarray comprising 1626 human recombinant proteins, selected in silico for being secreted or membrane associated. We screened sera from AIH patients on this microarray and compared the reactivity with that of sera from healthy donors and patients with chronic viral hepatitis C. We identified six human proteins that are specifically recognized by AIH sera. Serum reactivity to a combination of four of these autoantigens allows identification of AIH patients with high sensitivity (82%) and specificity (92%). Of the six autoantigens, the interleukin-4 (IL4) receptor fibronectin type III domain of the IL4 receptor (CD124), which is expressed on the surface of both lymphocytes and hepatocytes, showed the highest individual sensitivity and specificity for AIH. Remarkably, patients' sera inhibited STAT6 phosphorylation induced by IL4 binding to CD124, demonstrating that these autoantibodies are functional and suggesting that IL4 neutralization has a pathogenetic role in AIH.

Use of fresh-frozen plasma in 2012 at the Fondazione Ca' Granda Hospital of Milan: assessment of appropriateness using record linkage techniques applied to data routinely recorded in various hospital information systems.

BACKGROUND: The Quality Unit of a research and teaching hospital in Milan assessed the increased clinical use of fresh-frozen plasma in patients treated during 2012 in order to evaluate the appropriateness of this use. MATERIALS AND METHODS: For each patient in the study, a pathology profile was generated by means of record linkage techniques involving data collected through different information systems. Patients' information was combined using the patient identifier key generating pathology profiles exported to an Excel file. The profiles were reviewed by two haematologists who identified 101 potentially inappropriate treatments for which the medical records had to be reviewed manually. RESULTS: In 2012, 490 patients were transfused and for 473 cases the automatic record linkage provided a complete profile. The information relating to the remaining patients did not match, mainly because the patients underwent outpatient procedures for which clinical information is not automatically recorded. In the overall audit only 13 treatments were judged inappropriate. DISCUSSION: Our study supports the view that record linkage techniques applied to data routinely recorded in different hospital information systems could be potentially extended to support clinical audits, enabling the generation of automated patient profiles that can be easily evaluated, relegating manual checks on medical records to doubtful cases only. Moreover, the method applied in this study allows the analysis of a full set of cases instead of sample surveys, increasing the robustness of the audit results.

Risk of hepatocellular carcinoma in relation to ABO blood type.

BACKGROUND: Mortality and incidence rates of hepatocellular carcinoma (HCC) parallel the geographical distribution of hepatitis B and C viruses among the general population, however genetic factors modulate individual cancer risk. AIMS: ABO blood type, as a genetic marker, has previously been associated with the risk of several malignancies; we aimed to evaluate whether an association exists with HCC. METHODS: This is a retrospective case-control study based on ABO distribution in 194 patients with HCC, compared with 215 decompensated cirrhotics without HCC listed for liver transplantation, and 90,322 healthy blood donors. RESULTS: In patients with HCC, prevalence of blood type O was 35%, vs. 44% in cirrhotics (OR: 0.67, 95% CI 0.45-0.99; p=0.046) and 45% in blood donors (OR: 0.65, 95% CI 0.48-0.88; p=0.004). CONCLUSIONS: ABO blood type non-O is associated with higher risk of hepatocellular carcinoma, compared to cirrhotics without HCC and healthy subjects.